1. Field of the Invention
This invention relates to methods for measuring proteasome activity in biological samples. More particularly, the invention relates to methods for monitoring drug action following in vivo administration of a proteasome inhibitor.
2. Summary of the Related Art
The 26S proteasome is the multi-catalytic protease responsible for the majority of intracellular protein turnover in eukaryotic cells, including proteolytic degradation of damaged, oxidized or misfolded proteins, as well as processing or degradation of key regulatory proteins required for various cellular function (Ciechanover, Cell 79: 13-21 (1994); Coux et al., Ann. Rev. Biochem. 65:801-847 (1995); Goldberg et al., Chemistry and Biology 2:503-508 (1995)). Protein substrates are first marked for degradation by covalent conjugation to multiple molecules of a small protein, ubiquitin. The resultant polyubiquitinated protein is then recognized and degraded by the 26S proteasome.
Constituting the catalytic core of the 26S proteasome is the 20S proteasome, a multi-subunit complex of approximately 700 kDa molecular weight. Coux et al. (Ann. Rev. Biochem. 65:801-847 (1995)) teaches that the 20S proteasome does not by itself degrade ubiquitinated proteins, but does possess multiple peptidase activities. Based on substrate preferences, Coux et al. characterizes these activities as chymotrypsin-like, trypsin-like, post-glutamyl hydrolase, branched chain amino acid preferring, and small neutral amino acid preferring. Coux et al. also teaches that a dramatic activation of 20S proteasome activity can be induced by various in vitro treatments, such as heating to 55xc2x0 C., incubation with basic polypeptides, sodium dodecyl sulfate (SDS), guanidine HCI or fatty acids, dialysis against water, or by physiological regulators such as PA28 or PA700. McCormack et al. (Biochemistry 37:7792-7800 (1998)) teaches that a variety of peptide substrates, including Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Arg-AMC, and Z-Leu-Leu-Glu-2NA, wherein Suc is N-succinyl, AMC is 7-amino-4-methylcoumarin, and 2NA is 2-naphthylamine, are cleaved by the 20S proteasome.
The ubiquitin-proteasome pathway plays a central role in a large number of physiological processes. Deshaies (Trends in Cell Biol. 5: 428-434 (1995)) and Hoyt (Cell 91:149-151 (1997)) teach that regulated proteolysis of cell cycle proteins, including cyclins, cyclin-dependent kinase inhibitors, and tumor suppressor proteins, is required for controlled cell cycle progression and that proteolysis of these proteins occurs via the ubiquitin-proteasome pathway. Palombella et al., WO 95/25533 teaches that activation of the transcription factor NF-xcexaB, which itself plays a central role in the regulation of genes involved in the immune and inflammatory responses, is dependent upon the proteasome-mediated degradation of an inhibitory protein, IxcexaB-xcex1. Goldberg and Rock, WO 94/17816 discloses that the continual turnover of cellular proteins by the ubiquitin-proteasome pathway plays an essential role in antigen presentation.
While serving an essential physiological role, the ubiquitin-proteasome pathway also mediates the inappropriate or accelerated protein degradation that occurs as a result or cause of pathological conditions such as cancer, inflammatory diseases, or autoimmune diseases, in which these normal cellular processes have become deregulated. In addition, Goldberg (U.S. Pat. No. 5,340,736 (1994)) teaches that the cachexia or muscle wasting associated with conditions such as cancer, chronic infectious diseases, fever, muscle disuse (atrophy), nerve injury, renal failure, and hepatic failure results from an increase in proteolytic degradation by the ubiquitin-proteasome pathway. Gonzales et al. (J. Exp. Med. 184:1909 (1996)) teaches that the cytoskeletal reorganization that occurs during maturation of protozoan parasites is proteasome-dependent.
Inhibition of proteasome activity thus offers a promising new approach for therapeutic intervention in these and other conditions directly or indirectly mediated by the proteolytic function of the proteasome. Goldberg et al. (Chemistry and Biology 2:503-508 (1995)) teaches that proteasome inhibitors block the inflammatory response in vivo in animal models of human disease.
The present inventors are developing proteasome inhibitors for the treatment of inflammatory and autoimmune diseases and cancer. We have found that, when administering a proteasome inhibitor to a mammal, it is essential that the dose regimen be carefully selected so as to avoid excessive proteasome inhibition. Typically, dose regimens for new drug candidates are determined by measuring the concentration of the drug in a biological sample and setting the dose amount and dose frequency so as to achieve the desired drug level (see, e.g., Ritschel, Handbook of Basic Pharmacokinetics, Fourth Edition, Drug Intelligence Publications, Inc., Hamilton, Ill., 1992). The present inventors have discovered that these standard procedures are unsuitable for proteasome inhibitors. There is thus a need in the art for sensitive methods for monitoring proteasome inhibitor drug action.
The invention provides sensitive methods for monitoring proteasome inhibitor drug action. The present inventors have surprisingly discovered that ex vivo assay of proteasome activity, rather than drug concentration, in biological samples provides a useful method for monitoring pharmacodynamic drug action of proteasome inhibitors and that this data provides guidance for selecting a future dose amount and dose frequency of the proteasome inhibitor to be administered in the future.
In a first aspect, the invention provides a method for monitoring pharmacodynamic drug action of a proteasome inhibitor in a mammal, comprising administering the proteasome inhibitor to the mammal; obtaining one or more test biological samples from the mammal at one or more specified times after administering the proteasome inhibitor; measuring proteasome activity in the test biological sample or samples; determining the amount of proteasome activity in the test biological sample or samples; and comparing the amount of proteasome activity in the test biological sample to that in a reference biological sample obtained from a mammal to which no proteasome inhibitor has been administered.
In a second aspect, the invention provides a method for determining dose regimen for a proteasome inhibitor, comprising administering the proteasome inhibitor to the mammal; obtaining one or more test biological samples from the mammal at one or more specified times after administering the proteasome inhibitor; measuring proteasome activity in the test biological sample or samples; determining the amount of proteasome activity in the test biological sample or samples; comparing the amount of proteasome activity in the test biological sample to that in a reference biological sample obtained from a mammal to which no proteasome inhibitor has been administered; and selecting a dose amount and dose frequency of the proteasome inhibitor to be administered in the future.
In a third aspect, the invention provides a method for determining baseline proteasome activity in a mammal, including a human, comprising obtaining one or more biological samples from the mammal; measuring proteasome activity in the biological sample or samples; and determining the amount of proteasome activity in the biological sample or samples. In one preferred embodiment, the mammal suffers from a disease or pathological condition. In another preferred embodiment, the mammal has been administered a drug. In certain embodiments, the method further comprises determining a dose amount and dose frequency of a proteasome inhibitor to be administered to the mammal.
In a fourth aspect, the invention provides a kit for measuring proteasome activity in a biological sample from a mammal, the kit comprising means for preparation of the biological sample and means for measuring proteasome activity. In certain preferred embodiments, the mammal is a human. In certain other preferred embodiments, the biological sample is a blood, urine, or tissue biopsy sample.